The MHC Class I protein consists of an α-chain (α-1 to 3 and a transmembrane domain) and β2-microglobulin. It is polygenic (3 gene loci for MHC-class I protein in the haploid genome) giving rise to six different MHC class I protein α-chains (in humans two HLA-A, two HLA-B, two HLA-C). The MHC is further polymorphic. The human HLA-A allele A*0201 is prevalent in about 30% to 50% of the caucasian population (see e.g. Player, et al., J. Immunother. Emphasis Tumor Immunol. 19 (1996) 357-363).
Human cytomegalovirus huCMV (=human herpesvirus 5, HHV-5) is one of the largest human viruses. Its genome comprises around 230,000 bp linear double stranded DNA and encodes more than 160 proteins (see e.g. Davison, A J., et al., J. Gen. Virol. 84 (2003) 17-28).
The CMV has evolved to become a sublime parasite of the human genome and it is a potent immunogen and triggers strong immune responses from all arms of the immune system. This virus appears to be among the most immunodominant antigens known to the human immune system and stimulates CD8+-T-cell responses of unprecedented magnitude.
The CMV “latency” depends on chronic immune suppression of CMV viruses rather than a change in the pattern of viral transcription (see e.g. Moss & Khan, Human Immunology 65 (2004) 456-464).
CD8+-T-cell immune responses are not directed evenly against all CMV proteins but are focused. The CMV proteins pp65 and IE-1 are the predominant targets (see e.g. McLaughlin-Taylor, E., et al., J. Med. Virol. 43 (1994) 103-110; Moss & Khan, Human Immunology 65 (2004) 456-464).
The frequency of CMV-specific T-cells is very high with frequencies for individual peptides in the order of up to 1 to 2% of the total CD8+-T-cell repertoire (see e.g. Moss & Khan, Human Immunology supra; Wills, M. R., et al., J. Virol. 70 (1996) 7569-7579).
The CMV-specific CD8+-T-cell response increases markedly with age and individual HLA-peptide tetramers frequently stain in excess of 10% of the total CD8+-T-cell pool (see e.g. Khan, N., et al., J. Immunol. 169 (2002) 1984-1992).
The total CD8+-T-cell response in healthy elderly donors could constitute approximately 50% of the CD8+-T-cell repertoire.
The enormous CD8+-T-cell expansions are often very clonally restricted, and it is estimated that CMV is the cause of at least 30% of the clonal CD8+-T-cell expansions that are seen in peripheral blood with aging. The total CD8+-T-cell count is twice as high in CMV-seropositive donors older than age 60 years in comparison to a CMV-seronegative cohort (see e.g. Looney, R. J., et al., Clin. Immunol. 90 (1999) 213-219).
A fusion of soluble HLA and β-2-microglobulin is reported by Mottez et al. (Eur. J. Immunol. 21 (1991) 467-471); Godeau et al. (J. Biol. Chem. 267 (1992) 24223-24229) and Mage et al. (Proc. Natl. Acad. Sci. 89 (1992) 10658-10662). A fusion of viral-derived peptide with soluble HLA and β-2-microglobulin is reported by Mottez et al. (J. Exp. Med. 181 (1995) 493-502). A fusion of an immunoglobulin heavy chain with soluble HLA and co-expressed β-2-microglobulin is reported by Dal Porto et al. (Proc. Natl. Acad. Sci. USA 90 (1993) 6671-6675). A tetrameric multi-function protein of biotinylated peptide-soluble HLA and β-2-microglobulin with streptavidin chemically coupled to a Fab is described by Robert et al. (Eur. J. Immun. 30 (2000) 3165-3170). A chemically coupled Fab with a fusion of viral-derived peptide with soluble HLA and β-2-microglobulin is reported by Robert et al. (Cancer Immunity 1 (2001) 2). A fusion of a viral-derived peptide with soluble HLA and β-2-microglobulin to a murine monoclonal antibody heavy chain is reported by Greten et al. (J. Immunol. Methods 271 (2002) 125-135). An E. coli expression of scFv fusions without peptide, in vitro refolding and peptide loading is reported by Lev et al. (J. Immunol. 169 (2002) 2988-2996; Proc. Natl. Acad. Sci. 101 (2004) 9051-9056), and Novak et al. (Int. J. Cancer 120 (2006) 329-336). The use of biotinylated soluble MHC loaded with peptides and coupled to streptavidin fused Fab or scFv antibodies is reported by Mous et al. (Leukemia 20 (2006) 1096-1102).
In WO 2005/099361 are reported MHC class I—peptide-antibody conjugates with modified beta-2-microglobulin. Exemplary conjugates as reported in WO 2005/099361 are obtained by in vitro conjugation of the alpha chain of the MHC-multi-function protein (HLA) or by the co-expression from separate genes in the same cell.
In US 2004/0091488 antigenic constructs of major histocompatibility multi-function protein class I antigens with specific carrier molecules are reported. These reported fusion polypeptides lack the hinge region.
In WO 99/13095 the use of multivalent chimeric peptide-loaded MHC/IG molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses. Methods and pharmaceutical compositions for immune depletion, particularly useful in the treatment of cancer are reported in WO 02/102299. Oelke, M., et al. (Nat. Med. 9 (2003) 619-624) report the ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig coated artificial antigen presenting cells. The removal of target cells by circulating virus-specific cytotoxic T-cells using MHC class I comprising complexes is reported in WO 2012/175508. Robert, B., et al. (Eur. J. Immunol. 30 (2000) 3165-3170) report that antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.